The promise of skeletal regenerative medicine to mitigate age-related bone loss and support fracture healing critically depends on the ability to manipulate the biological properties of osteogenic progenitor cells. Transcriptional and post-transcriptional regulatory mechanisms control cell fate determination and phenotype- specific gene expression when progenitor cells commit to the osteoblast lineage. MicroRNAs (miRNAs or miRs) and epigenetic regulators (EpiRegs) regulate osteogenic differentiation by controlling the expression of TFs. Therefore, this proposal will focus on miR-TF-EpiReg circuits that control osteoblast differentiation. The central hypothesis of the overall project is that osteoblast differentiation is controlled by miRNAs- TF-EpiReg circuits. Based on recent preliminary data, this proposal has evolved to address the specific working model that miR155 suppresses while miR101 stimulates osteoblast maturation by targeting distinct TFs and EpiRegs, respectively. Maximal expression of miR155 occurs in undifferentiated osteoblasts where we predict that miR155 - together with co-regulated miRNAs - controls a group of common TF targets that attenuate osteoblast maturation. For miR101, we observed that it is maximal in mature osteoblasts and targets a critical epigenetic regulator, Ezh2, which methylates histone H3. Our preliminary data show that Ezh2 expression is inversely regulated with miR101 and is critical for normal skeletal development, based on our phenotypic characterization of a conditional Ezh2 null mouse model. We will determine (i) the biological significance of miR155 in a novel miR/TF circuit that suppresses osteoblast differentiation in cell culture and in transgenic mouse models that conditionally express miR155 (Aim 1), as well as (ii) the importance of a miR101-Ezh2 axis during osteoblast differentiation in vivo and ex vivo by conditional expression of miR101 in transgenic mice (Aim 2). Upon completion of the primary objectives of this proposal, my long term goals are to examine the molecular consequences of miRNA dependent TF-EpiReg networks using RNASeq and ChiP-Seq approaches to define new drug-sensitive regulatory pathways. These studies should ultimately lead to the development of osteotropic drugs that could become new bone anabolic therapies. This future work as independent investigator would leverage my combined expertise in pharmacology, epigenetics and mouse bone biology that I acquired during doctoral and post-doctoral training.